Nucleotide excision repair (NER) is a highly conserved pathway from bacteria to humans that removes a wide variety of DNA lesions caused by environmental agents such as UV light and air pollutants. In addition, NER is important for the removal of adducts induced by several anticancer drugs, such as cisplatin. One of the fundamental questions in the field of DNA repair is how a modest number of repair proteins scan through several million (for bacteria) to a few billion base-pairs (for mammalian cells) of non-damaged DNA to find rare damaged bases. This project combines single molecule approaches (atomic force microscopy, and oblique angle fluorescence) with biochemical approaches to examine how bacterial and eukaryotic nucleotide excision repair proteins detect and remove damaged nucleotides from DNA. This study uses a novel optical platform for viewing single molecules in real-time moving on DNA and will give a dynamic view of how these protein machines assemble on DNA and track down DNA lesions. This highly innovative project has three main aims: 1) to investigate how bacterial NER proteins achieve highly specific recognition and repair of DNA damage; 2) to characterize the search mechanisms employed by human damage recognition proteins, XPC-HR23B, XPA, RPA, and UV-DDB; and 3) to examine the dynamics of human XPD (ERCC2), and XPB (ERCC3) helicase proteins on DNA. This project will test the hypothesis that the bacterial and human NER proteins share similar modes of DNA binding and searching mechanisms for damage detection and processing. Completion of these aims will help to revolutionize the field of DNA repair by developing new imaging techniques that allow direct visualization and real-time measurements of protein complexes in all stages of repair. They will also begin to address how damage is detected in the context of chromatin. In future years, they will also lay the ground work for imaging single-molecules in real time in living cells.